Tagging and recovery of elements associated with target molecules

ABSTRACT

The invention provides a method for identifying elements associated with a target molecule comprising the steps of: (a) providing a probe capable of binding by specific molecular interaction to a predetermined specifically defined region of a target molecule, the probe associated with or capable of recruiting an enzyme; (b) adding a tag capable of being activated by the enzyme such that it can attach to elements in the vicinity of the enzyme; and (c) isolating elements having the tag attached thereto, wherein the defined region occurs once, twice, or in a low number of copies in the target molecule. Preferably the tag can attach only to elements in the vicinity of the enzyme.

The present invention relates to a new method for identifying elementsassociated with target molecules.

Many genes and gene clusters are controlled by known (or unknown)distant regulatory elements that are necessary for high-levelexpression. Identification of these regulatory elements is an expensiveand time-consuming process. Previous attempts to identify such distantregulatory elements have used a number of different methods, but mostdirectly by scanning large genomic regions for DNase I hypersensitivitysites, followed by functional analysis of those regions linked toreporter genes in transgenic mice. This method of identification willclearly take a very long time.

The beta-globin locus is the prototypical gene cluster regulated bydistant regulatory elements; the search for the beta-globin regulatoryelements took approximately 10 years. Experiments designed to locate thebeta-globin gene regulatory elements began in the late 1970s. In theearly 1980s data arose that suggested distant elements were involved. Athalassemia patient was studied whose genome contained an intactbeta-globin gene but a large deletion upstream of the gene. This lead tothe conclusion that a distant upstream element must be involved in theregulation of the gene (Kioussis et al., 1983). Indeed, transgenescontaining the beta-globin gene alone achieve only very low levels ofexpression at best (Townes et al., 1985) In 1985 a series of DNase Ihypersensitive sites were mapped 40-60 Kb upstream of the beta-globingene (Tuan et al., 1985). In 1987 it was finally shown that thishypersensitive site region, collectively known as the locus controlregion (LCR), was sufficient to induce high level, position independent,copy number dependent gene expression when linked to the beta-globingene (Grosveld et al., 1987). Defects in human beta-globin geneexpression, or hemoglobinopathies, are the most common genetic diseasesworldwide. The ability to induce high-level expression of anartificially introduced beta-globin gene is therefore of significanttherapeutic use. In addition, the ability to locate control regions ofother genes is clearly desirable.

Chromatin conformation capture (3C; Decker et al 2002) has been used todetermine the conformation of a yeast chromosome to try to determine theinteraction of genes and control regions. However, many technicalproblems arise when trying to apply this method to higher eukaryotes,not least because the mammalian genome is approximately 200 times thesize of a yeast genome. The 3C has several disadvantages: 3C does notenable recovery of in situ labelled molecules, nor does 3C give a veryhigh degree of resolution. In addition, other disadvantages of the 3Ctechnique result because this technique allows only an averageconformation of a chromosome to be calculated; this means that if allthe cells used in the technique are not homogeneous or the molecularconformation is dynamic, specific interactions may be overlooked.Further, the 3C technique does not provide a method for determiningwhich proteins or other molecules are associated with the genome.

Fluorescence in situ hybridisation (FISH) is a previously knowntechniques which uses hapten-labelled nucleotide probes followed byanti-hapten antibodies conjugated to fluorophores to determine the siteof an actively transcribed gene via the antibody's ability tospecifically bind to the hapten. Covalent tag deposition has commonlybeen used to enhance the signals obtained using the above technique.Kits enabling performance of covalent tag deposition to enhance signalsare obtainable from NEN Dupont and are called TSA™ (Tyramide SignalAmplification™). However, this technique has not provided means forpurifying molecular complexes from specific sites or in the immediatevicinity of specific sites in or on cells. Neither FISH nor TSA allowfor detection (and thus identification) of, for example, the interactionof distant regulatory elements with an actively transcribed gene. Thereis no technique presently available to use for detecting(and thusidentifying) the interaction of distant regulatory elements with anactively transcribed gene during the time of transcription.

Techniques are known which can be used for identification and analysisof proteins involved in protein complexes. ImmunoPrecipitation (IP) ismost commonly used to ‘pull down’ proteins associated in a complex witha target protein(s). However no techniques exist to analyse, forinstance, molecules or complexes which are only involved in “loose”functional interactions with another complex or which only function inthe vicinity of another protein.

van Steensel et al(Nature Genetics, 27, 304-308, 2001) describe a methodof genome-wide Chromatin profiling using targeted DNA adeninemethyltransferase (DAM). A “GAGA factor” (GAF) conjugate with DAM bindspredominantly to the motif GAGA, which motif is present in numerouseuchromatic sites in chromosomes. This provided a large-scale techniquefor mapping of protein-binding sites in the genome of Drosophilia.Because methylation by tethered DAM spreads over 2-5 kb from a discreteprotein binding sequence, target locus may be mapped with a resolutionof a few kilobases.

According to the present invention there is provided a method foridentifying elements associated with a target molecule comprising thesteps of:

-   -   (a) providing a probe capable of binding by specific molecular        interaction to a predetermined specifically defined region of a        target molecule, the probe associated with or capable of        recruiting an enzyme;    -   (b) adding a tag capable of being activated by the enzyme such        that it can attach to elements in the vicinity of the enzyme;        and    -   (c) isolating elements having the tag attached thereto,

wherein the defined region occurs once, twice, or in a low number ofcopies in the target molecule.

According to the invention it may be preferable that the tag can attachonly to elements in the vicinity of the enzyme.

Further, according to the invention it may be that the “low copy number”of the defined region of the target molecule is selected from the groupof integral numbers of more than 2 up to 1000.

The target molecules may include RNA molecules, DNA molecules, proteinsor peptides, lipids, or other, artificial compounds.

The method of the invention differs significantly from that of vanSteensel et al. Their method is used to modify DNA on a genome widescale. By fusing the DAM methylase to a DNA-binding or chromatinprotein, they aim to methylate DNA wherever the fusion protein interactswith genomic sequences. This may be hundreds to several tens ofthousands (or even millions) of sites within an individual cells genome.They then recover a highly heterogenous, complex mixture of DNAmolecules from an unknown number of unrelated genomic sites. The methodof the invention on the other hand can be targeted to a single gene orDNA locus. Only genomic DNA sites in the immediate vicinity, or incontact with, the target locus are labelled and thus a much morespecific mix of DNA molecules can be recovered. The van Steensel methodis broadly targeted to a number of sites but the targets are unknown andunrelated. The method of the invention can specifically target a singlesite or sites, along with elements involved in functional interactionswith that site.

It is a particular advantage of the present invention that it provides amethod of using the precise targeting power of specific molecularinteractions such as in situ hybridization or immunohistochemistry tobind a probe just to a specific or unique region of a target moleculesuch as a complementary DNA, genomic locus, RNA species, or a protein orlipid cellular structure, the probe associated with or capable ofrecruiting an enzyme. This allows tagging of elements associated with,and only in the vicinity of, that region of the target molecule.

When the target is RNA, the elements which may be associated with thesetarget molecules and which may be identified (or whose mode of actioncan be understood) by using the technique of the present inventioninclude: distant regulatory elements (i.e. DNA elements via theirchromatin protein association) that are in proximity to the RNA of anactively transcribed gene; RNA binding proteins such as those involvedin RNA processing or stabilization/regulation/etc; proteins and proteincomplexes which facilitate the interactions between regulatory elementsand a gene; proteins and protein complexes involved in the activation ofgenes; proteins and protein complexes involved in the regulation ofchromatin structure in and around active genes; and transcriptionfactors.

When the target is DNA, the elements which may be associated with thesetarget molecules and which may be identified (or whose mode of actioncan be understood) by using the technique of the present inventioninclude: distant regulatory elements (i.e. DNA elements via theirchromatin protein association) that are in proximity to the targetedDNA; other DNA elements in proximity to the targeted DNA, which may befor example, engaged in functional interactions with the target sequence(e.g. boundaries, insulators, structural or architectural interactions);analysis of higher order chromatin structure, for example the analysisof tertiary chromatin interactions (chromatin folding); mappingchromatin interactions in entire loci or whole genomes (with the aid ofhigh throughput technology); protein/protein complexes involved inregulation of gene expression or the control of chromatin structure.

When the target is protein, the elements which may be associated withthese target molecules and which may be identified (or whose mode ofaction can be understood) by using the technique of the presentinvention include: DNA elements in proximity to a protein; RNA moleculesin proximity to a protein; or other proteins/protein complexes bound to,or in the vicinity of a targeted protein (e.g. identifying other proteincomponents of the LCR-beta-globin gene complex at different stages ofdevelopment, or identifying the in-vivo ligands of a specific receptor-or vice versa).

When the target is lipid, the elements which may be associated withthese target molecules and which may be identified (or whose mode ofaction can be understood) by using the technique of the presentinvention include: DNA elements in proximity to a lipid or artificialcompound RNA molecules in proximity to a lipid or artificial compound;or proteins/protein complexes bound to, or in the vicinity of a targetedlipid or artificial compound.

The probe usable in the present invention may be a DNA probe, an RNAprobe or an antibody specific for a protein, lipid or other molecule.

The probes used can be associated with the enzyme throughantibody/enzyme conjugates, or enzyme/target molecule fusion.

The method by which the enzyme may be targeted to a specific moleculemay be varied depending on the molecule to be targeted. For example,using a labelled probe specific for a DNA molecule, usingimmuno-histochemistry, or using a fusion of a protein (or other moleculeof interest) and the enzyme. Preferably antibody/enzyme conjugates maybe used. In one preferred embodiment, when the target molecule is RNA, ahapten-labelled probe specific to the intron of an active gene can beadded, followed by addition of a hapten-specific Fab fragment/enzymeconjugate. One hapten which may be used is digoxygenin (DIG); othersinclude biotin, dinitriphenol and FITC.

An enzyme which may be used in the present invention is Horse RadishPeroxidase. This enzyme can be used in combination with a tyramidemolecule such as biotin-tyramide, dinitrophenol-tyramide orFITC-tyramide. These molecules form highly reactive, short-livedreactive radicals when catalysed by an enzyme, which bind to electrondense amino acids. As a result of their highly reactive nature, theyonly bind to amino acids in the immediate spatial vicinity. FIG. 12shows a pronounced peak in the b1 and b2 loci, over a distance of 20-25kb. The extent of the spread of these highly reactive radicals may beprecisely controlled by varying the reaction conditions. This can resultin a precise targeting method.

Another enzyme/TAG combination is ubiquitin-conjugating enzyme, withubiquitin as a tag. Protein kinase could also be used as the enzyme(there are several with varied specificities) with phosphate as a tag.In this example a kinase which is able to add a phosphate to anucleosomal protein (if looking for chromatin tagging) or other proteinof interest should be used. Antibodies against the specifically modifiedepitope of the particular amino acid residue receiving the phosphatecould be used to target isolate the tagged elements.

DNA Adenine Methyltransferase (DAM) is another enzyme which could beused, with a methyl group as the tag. In a slight variation of theprocedure, instead of using a tag to pull out the labelled material onecould use a restriction enzyme that will cut only DNA which isspecifically methylated by DAM. DAM adds a methyl group to the adeninein the sequence GATC. This methylated site can only be cut by the DNArestriction endonuclease DpnI. DAM is normally only found in bacteriasuch as E. coli so it could be used in eukaryotic cells without anyinterference from endogenous methyltransferases which only methylateother sequence combinations. With this method no affinity chromatographyis required. We would simply purify the DNA from the DAM treated cellsand cut with DpnI and then isolate small DNA fragments that are releasedfrom the mixture of genomic DNA can be isolated. Careful selection ofthe target is preferred to prevent the DAM methylating sections of DNA,not in the immediate spatial vicinity of the interaction being studied.The small sites released by DpnI digestion can then be labelled withradioisotopes, etc., and used for diagnostic hybridization to amicroarray, for example (van Steensel et al 2001).

Other enzyme/tag combinations could be used: any enzyme which canactivate a tag molecule to deposit onto another molecule, for exampleprotein, DNA, RNA, lipid etc in a manner such that the tagged productcan then be isolated by whatever means (eg. affinity chromatography orimmunoprecipitation) can be used in this technique.

Before separation, the molecules which have been tagged can be disruptedinto smaller fragments using, for example, sonication, enzymaticcleaving, shearing with a French Press or small bore syringe, or anothermethod which achieves such a result.

Analysis of the DNA obtained using the above method can be used toidentify any regulatory elements which were in proximity to the activegene, because these elements become labelled with the tag, due to theirproximity to the site HRP activity. The DNA can then be analysed by anumber of quantitative techniques, for example Quantitative PCR (forexample Real-Time PCR (Wittwer et al., 1997)) or semi-quantitative PCR,slot blot or microarray (Granjeaud et al., 1999), among others. Thisanalysis allows scanning, high-throughput, high resolution analysis ofany gene locus for hundreds or thousands of kilobases in eitherdirection.

An embodiment of the present invention will now be described in moredetail, by way of example, with reference to the drawings, in which:

FIG. 1 is a schematic diagram showing a transcriptionally active gene invivo. RNA polymerase II (open circles) transcribes a chromosomal gene ornucleosomal DNA template (DNA represented by curved lines wrapped aroundnucleosomes, (cylinders)). The RNA polymerase produces a nascent RNAprimary transcript (diagonal straight lines).

FIG. 2 is a schematic diagram showing in situ hybridisation. Acomplementary oligonucleotide probe is hybridised to the intron of thenascent RNA transcript. The oligonucleotide probe is labelled with ahapten, in this case digoxygenin (diamond).

FIG. 3 is a schematic diagram showing immunological detection of haptenprobe. An anti-digoxygenin antibody (black oval) conjugated tohorse-radish peroxidase enzyme (triangle) is added. Theantibody/peroxidase complex binds to the digoxygenin labelled,oligonucleotide probe.

FIG. 4 is a schematic diagram showing the addition of biotin tyramide.Biotin-tyramide consists of a biotin molecule (B) linked to aphenol-like, tyramide chemical ring (hexagon with circle). When thetryamide comes in contact with the peroxidase, the tyramide is convertedto a short-lived, highly reactive radical which is capable of immediatecovalent attachment to electron dense moieties of nearby proteins.

FIG. 5 is a schematic diagram showing the labelling of chromatinproteins in the immediate spatial vicinity. Biotin-tyramide depositioncan also occur on chromatin proteins of sequences which are in theimmediate vicinity. Such as, enhancers, locus control regions or othergene regulatory elements. DNA bound transcription factor (large oval).

FIG. 6 is a schematic diagram showing the disruption of the chromatin.Chromatin is disrupted via sonication or some other method.

FIG. 7 is a schematic diagram showing purification of elements byaffinity chromatography. Biotinylated protein/DNA complexes are purifiedby affinity chromatography with a strepavidin column.

FIG. 8 is a schematic diagram showing cross link reversal. Theformaldehyde chemical cross-links are reversed and DNA and/or proteinsare purified for analysis.

FIG. 9 is a schematic diagram showing the mouse beta-globin locus(genes=black boxes) and locus control region (LCR) and illustrates onemodel of LCR action: action at a distance.

FIG. 10 is a schematic diagram showing the mouse beta-globin locus andlocus control region (LCR) and illustrates another model of LCR action:direct LCR-gene interaction.

FIG. 11 is an image of a typical cell after visualisation of thespecifically targeted biotin tyramide deposition.

FIG. 12 is a graph showing the results of Quantitative real-time PCRanalyses of βmaj-directed RNA TRAP showing various sequences in the βglobin locus and neighbouring olfactory receptor gene locus.

FIG. 13 is a graph showing the results of βmin-directed RNA TRAPassaying various sequences in the β globin locus and neighbouringolfactory receptor gene locus.

FIG. 14 is a schematic diagram showing the hypothesised interaction ofthe mouse beta-globin gene and locus control region (LCR).

Many genes and gene clusters are thought to be regulated by distantregulatory elements, which may be located tens to hundreds of kilobasesaway. The best characterised example of a distant element regulating acluster of genes is the beta-globin locus control region (LCR), shown inFIG. 9. The LCR consists of a series of DNase I hypersensitive sites(HS) (1 to 6). At the core of each HS is a 200-300 bp region which ispacked with transcription factor binding sites. The LCR is absolutelyrequired for high level transcriptional activation of all thebeta-globin genes. Two models have been proposed to explain the actionof the LCR, although no direct proof exists for either mode of action,these are shown in FIGS. 9 and 10. The first model (FIG. 9) proposesthat the LCR works at a distance. The LCR creates a large region of openchromatin surrounding the genes and recruits and sends factors necessaryfor gene activity along the chromatin. The second model (FIG. 10)proposes that the LCR physically contacts the gene(s) through long rangechromatin interactions, essentially looping out the interveningsequences and activating transcription directly.

To determine if an actively transcribed beta-globin gene is in directphysical contact with the distant (40 Kb) LCR in vivo, the followingtechnique was used (see FIGS. 1-8). Firstly, fetal liver, the main siteof erythropoiesis in the developing foetus, is taken and disrupted, andthe cells are spread in a monolayer on a slide, prior to cross-linkingwith formaldehyde. In situ hybridization is performed using adigoxygenin (DIG)-labelled oligonucleotide probe (FIG. 2), specific forthe intron of the mouse beta-major globin gene. The enzyme Horse RadishPeroxidase (HRP) is then targeted to an RNA molecule using an anti-DIGantibody conjugated to Horse Radish Peroxidase (HRP) (FIG. 3), thuspinpointing HRP enzyme activity to the site of the actively transcribedgene.

Next, biotin-tyramide (FIG. 4) is added as a molecular tag; it isactivated by the HRP to cause it to covalently attach to electron denseamino-acids in the immediate vicinity. After the tag is covalentlyattached (FIG. 5), the cells are sonicated to give small, solublechromatin fragments (FIG. 6) having an average DNA size of 400 bp. Thebiotinylated chromatin is then purified using streptavidin agaroseaffinity chromatography (FIG. 7), cross-links are reversed and the DNAis purified. Multiple amplicons across the locus can then be analysedusing quantitative or semi-quantitative PCR and/or slot blotting.

By using the above technique on the mouse beta-globin gene locus, it wasfound that high-level expression of the beta-globin genes is totallydependent on an extensively characterised, distal, regulatory elementknown as the LCR. The LCR and active beta-major gene are found to be insignificant proximity in the mouse beta-globin locus in vivo; HS2appears to be in intimate contact with the beta-major gene, and the twoactive adult genes also appear to be in close proximity (FIG. 3).

EXAMPLES Example 1

RNA FISH-TRAP

E14.5d fetal livers from balb/c mice, in which only the adult-type b-majand b-min genes are expressed, were disrupted in ice-cold PBS. The cellswere spread on poly-L-lysine coated slides and fixed in 4% formaldehyde,5% acetic acid for 18 minutes at room temperature. Subsequentslide-washing, permeabilization, probe-hybridisation, and posthybridisation washing were performed as described in Gribnau, J. et al.(1998); the probes used being directed to intron 2 near the 3′ ends ofthe mouse b-maj globin primary transcript. Endogenous peroxidases werequenched in 0.5% H₂O₂ (in PBS) for 10 minutes followed by washing (5min) in TST (Tris, saline, Tween; 100 mM Tris ph7.5, 150 mMNaCl, 0.05%Tween 20) and blocking as described. Slides were then incubated with1:100 dilution of anti-DIG fab fragment/HRP conjugate for 45 minutes atroom temperature in a humidified chamber, washed twice (5 min each) inTST and then incubated for 1 minute with 1:150 biotin tyramide (NEN)under coverslips at room temp. The slides were then quenched again in0.5% H₂O₂ (in PBS) for 10 minutes, washed twice in TST (5 min) andtransferred to PBS ready for scraping. One of the slides was stainedwith an Avidin/Texas red conjugate for 45 minutes at room temperature.This slide was then washed, dehydrated, mounted and visualised asdescribed in Gribnau, J. et al. (1998)

Cells were scraped from the remaining slides; typically approximately 25million cells were recovered. The cells were spun down at 2900 g for 25minutes, resuspended in 2M NaCl, 5M Urea, 10 mM EDTA, and sonicated for200 seconds on ice (eight 25-second bursts with 1.5 minutes betweenbursts) using a Microson Ultrasonic cell Disruptor set at level 5. Crudechromatin was centrifuged for 15 minutes at 10,000 g, the supernatantcontaining the soluble chromatin was removed and the insoluble pelletwas resuspended in 2M NaCl, 5M Urea, 10 mL EDTA, and sonicated again.The suspension was centrifuged again and the two soluble fractions werecombined and dialysed overnight at 4° C. against PBS. This methodroutinely yielded chromatin fragments with an average DNA size of around400 bp.

10% of the soluble chromatin was set aside as the input and the rest waspassed over a streptavidin-agarose (Molecular Probes) affinity column.After binding, the column was washed with 3×700 μl PBS, 2×500 μl TSE 150(20 mM Tris pH8.0, 1% Triton, 0.1% SDS, 2 mM EDTA, 150 mM NaCl), 2×500μl TSE 500 (20 mM Tris pH8.0, 1% Triton, 0.1% SDS, 2 mM EDTA, 150 mMNaCl), and 3×700 μl PBS. The beads were then removed from the column,formaldehyde cross-links reversed and protein components digested byovernight incubation at 65° C. with 200 ug/ml proteinase K while shakingvigorously. The samples were treated with 20 Vg/ml RNase A for 30 min at37° C., 200 μg/ml proteinase K for 5 hours at 37° C., phenol-extractedand ethanol-precipitated using 20 mg/ml glycogen as carrier. DNA fromthe input (IP) fraction was quantified using a standardspectrophotometer. DNA concentration of the affinity purified (AP)fraction was measured by picogren quantification using IP as a standard.

Example 2

Real-Time PCR

Real-time PCR was performed with an ABI PRISM 7700 sequence detectorusing 2× SYBR green PCR master mix (Applied biosystems). For each primerpair a standard curve was generated using 30 ng, 5 ng, and 1 ng of IPwhich was then used to quantify the enrichment of 1 ng of AP (allreactions were performed in duplicate). All PCR products were run on a2% agorose gel to ensure all reactions gave a single product.

Enrichment of various sequences across the β-globin locus and alsoacross the neighbouring olfactory receptor gene (org), were measuredusing quantative real-time PCR. The measurements showed a 20-folded peakof enrichment near the transcription termination site of the b-maj gene,consistent with the position of the probes (FIG. 12). Enrichment droppedoff sharply upstream of the b-maj gene for over 25 kb in the area of thedevelopmentally silenced εy and βH1 genes, which are only sightlyincreased over background.

Strikingly, a peak of enrichment was observed over HS2, and to a lesserextent HS1 and HS3 of the LCR. This indicates these sites are in closeassociation with the active gene.

The fact that other HS in the LCR (HS4, 5 and 6) and the downstream3′HS1 (which is closer in base pairs to the βmaj gene than HS2) are notsignificantly enriched suggests they are outside the area of labellingand therefore not intimately associated with the active βmaj gene.Moreover, the low level of enrichment of these sites shows that there isno preferential labelling of areas of hypersensitive or open chromatin.To completely discount the possibility that these results were caused bya bias of biotin deposition in certain areas (e.g. open or hyperacetylated chromatin) a control random TRAP experiment was designed andperformed. By omitting the intron probe during the FISH-stage, biotindeposition becomes random across the genome and therefore any bias forcertain sequences would become apparent in the analysis of the APmaterial. There was no preferential selection for any of the sequencesin the globin locus, thus verifying that enrichment of HS2 in theβmaj-directed TRAP experiment is due to proximity to the active βmajgene and is not a chromatin bias. Repetition of the βmaj RNA TRAP assaythree times obtained similar results. DNA from one of the βmaj RNA TRAPassays was analysed by slot blot with multiple probes yielding similarresults. The data of this experiment provide the first direct evidencethat a distal enhancer is held in significant physical proximity to anactive gene that it regulates in vivo.

To distinguish between a co-transcriptional model in which both genesshare the LCR simultaneously or an alternating model in which the LCR isinvolved exclusively with a single active gene. RNA-TRAP was repeatedusing intron probes to the βmin gene located approximately 15 kbdownstream of βmaj. The results of this showed that HS2 is highlyenriched in the βmin-directed AP chromatin, indicating it is tightlyassociated with the active βmin gene (FIG. 13). In addition, HS4 of theLCR was significantly enriched over background levels and when comparedto HS1, 3, 5 and 6 of the LCR. The high level of enrichment of HS2 inboth the βmin and βmaj directed RNA-TRAP assays indicates it is tightlyassociated with the active gene for most of the time primary transcriptis present. The fact that βmaj-TRAP does not bring down the βmin geneand vice versa indicates the two genes are not closely associated.

There are many applications for the technique of the present invention,which can be performed in vivo, ex vivo, or in vitro.

One example of such a use is in transgenic animal technology: transgenicanimals are presently being used by a number of laboratory around theworld as bioreactors to produce large amounts of proteins of interest.The most commonly used method is to express the protein of interest inmilk under control of a highly expressed milk protein gene promoter.Most transgenic animals created with such a construct would not expressthe protein or express it at very low levels making them unusable. Sometransgenic animals may, by virtue of position effects at the site ofintegration of the construct, express larger amounts of the protein ofinterest. The addition of milk protein gene LCR-like sequences to theexpression construct would increase the number of transgenic animalswhich express the gene to 100% and increase the average level ofexpression in every animal. This would significantly decrease the costof production and greatly increase the yield.

When RNA is the target molecule, the method of the present inventionlabels only the cells in the population that are actively transcribingthe gene of interest. The advantage of this is specifically interactingsequences are highly enriched upon affinity chromatography, whether thepopulation is heterogeneous or the interaction is dynamic (Wijgerde etal., 1995). Another advantage of the present invention when RNA is thetarget molecule is this technique can detect (and thus identify) theinteraction of distant regulatory elements with an actively transcribedgene during the time of transcription. There is no other technique weknow of which can be used for this purpose. This technique canspecifically label and recover proteins at the site of transcription ina dynamic or heterogeneous population of cells and identify specificinteractions.

Another advantage of the present invention which results whatever thetarget molecule is, is the possibility of labelling and recoveringcomplexes in the vicinity of a target complex (as opposed to moleculeswhich are in direct interaction). The resultant enriched proteins couldbe analysed by a number of protein chemistry techniques such as Westernblotting, Mass Spectroscopy, fractionation, purification, polyacrylamidegel electrophoresis, etc.

The present invention provides a relatively easy and rapid method whichcan detect interactions between an actively transcribed gene and distantregulatory element(s). The technique can also be used to identify anysequence element involved in an interaction with any other targetsequence in vivo by virtue of their proximity.

The present invention provides a new way to identify the regulatoryelements involved in the activation of genes in a rapid and relativelyinexpensive way. It has also been used to address the question of howLCRs or enhancer elements function and in fact has provided the firstdirect evidence that the LCR functions by physically interacting with anactively transcribed gene in the beta-globin locus.

Data with RNA FISH shows that the method of the invention has clearlyidentified HS2 of the beta-globin locus control region. HS2 has beenshown previously through functional studies to be major, classicalenhancer element of the locus control region that drives beta-globingene expression in vivo. Therefore in similar experiments with othergenes the major enhancer element(s) driving those genes could beidentified by this technique. Function and/or industrial applications ofthe isolated elements could be inferred.

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1. A method for identifying elements associated with a target moleculecomprising the steps of: (a) providing a probe capable of binding byspecific molecular interaction to a predetermined specifically definedregion of a target molecule, the probe associated with or capable ofrecruiting an enzyme; (b) adding a tag capable of being activated by theenzyme such that it can attach to elements in the vicinity of theenzyme; and (c) isolating elements having the tag attached thereto,wherein the defined region occurs once, twice, or in a low number ofcopies in the target molecule.
 2. A method according to claim 1 whereinthe tag can attach only to elements in the vicinity of the enzyme.
 3. Amethod according to claim 1 wherein the low copy number of the definedregion of the target molecule is selected from the group of integralnumbers of more than 2 up to
 1000. 4. A method according to claim 1, inwhich the target molecule is selected from the group consisting of RNAmolecules, and DNA molecules.
 5. A method according to claim 1, in whichthe target molecule is selected from the group consisting of proteins orpeptides, lipids, or other, artificial compounds.
 6. A method accordingto claim 1 in which the elements which may be associated with the targetmolecule include distant regulatory elements, RNA, DNA, proteins andprotein complexes, transcription factors, or in-vivo ligands of aspecific receptor.
 7. A method according to claim 4 in which the probeis selected from the group consisting of DNA probe, and an RNA probe. 8.A method according to claim 5 in which the probe is selected from thegroup consisting of an antibody specific for a protein, lipid or othermolecule.
 9. A method according to claim 1 in which the probe isassociated with the enzyme through an antibody/enzyme conjugate, orenzyme/target molecule fusion.
 10. The method according to claim 1 inwhich the enzyme is targeted using a hapten labelled probe and then ahapten-specific Fab fragment-enzyme conjugate is added.
 11. The methodaccording to claim 1 in which the enzyme is targeted to RNA using ahapten-labelled probe specific to the RNA of an intron of an activegene, and then a hapten-specific Fab fragment/enzyme conjugate is added.12. The method according to claim 10 in which the hapten is dioxygenin,biotin, dinitrophenol or FITC.
 13. The method according to claim 1 inwhich the enzyme is Horse Radish Peroxidase and the tag isbiotin-tyramide.
 14. The method according to claim 1 in which elementsare isolated using affinity chromatography or ImmunoPrecipitation.
 15. Amethod for identifying elements of chromatin associated withtranscribing RNA comprising the steps of: (a) providing ahapten-labelled probe capable of binding by specific molecularinteraction to a predetermined specifically defined region of RNA of agene, (b) providing an antibody conjugated with the enzyme horse-radishperoxidase, the antibody being specific for the hapten; (c) addingbiotin-tyramide by such that it can attach to elements in the vicinityof the enzyme; (d) disrupting the chromatin; and (e) isolating elementsof chromatin having biotin attached thereto using affinitychromatography and purifying the elements.
 16. The method according toclaim 15 wherein in step (c) the tag can attach only to elements in thevicinity of the enzyme.
 17. The method of claim 15 in which thechromatin is disrupted using sonication, enzymatic cleaving, or shearingwith a French Press or small bore syringe.
 18. The method according toclaim 15 in which the hapten is digoxygenin.
 19. Elements isolated bythe method of any preceding claim
 1. 20. A method for identifying DNAassociated with a target molecule comprising the steps of: (a) providinga probe capable of binding by specific molecular interaction to apredetermined specifically defined region of a target molecule, theprobe associated with an DNA Adenine Methyltransferase; (b) adding arestriction enzyme that will cut only DNA specifically methylated byDAM; (c) isolating DNA cut by the restriction enzyme; and (d)identifying the isolated DNA.
 21. The method according to claim 20wherein the isolated DNA is analysed/identified using QuantitativeReal-Time PCR, slot blot or microarray.
 22. A method for conducting adrug discovery business, comprising: (i) by the method of claim 1,identifying DNA and/or protein associated with regulating geneexpression; (ii) generating a drug screening assay for identifyingagents which inhibit or potentiate regulation of gene expression by theDNA and/or protein identified in step (i); (iii) conducting animaltoxicity profiles on an agent identified in step (ii), or an analoguethereof; (iv) manufacturing a pharmaceutical preparation of an agenthaving a suitable animal toxicity profile; and (v) marketing thepharmaceutical preparation to healthcare providers.
 23. A method forconducting a bioinformatics business, comprising: (i) by the method ofclaim 1, identifying DNA and/or protein associated with a gene at achromosome location under a given condition; and repeating step (i);thereby (ii) generating a database comprising information identifyingdifferent DNA and/or protein associated with one or more genes under oneor more conditions.